Regulation of gene expression underlies many fundamental biological processes, including development of an organism from a fertilized egg, the response of organisms to extra-cellular signals, and the ingraining of memories. Very often aberrant gene regulation leads to diseased states, including cancer. This grant requests funds to pursue analyses of fundamental mechanisms of gene regulation that will help us understand the normal processes and shed light on how they can go awry. The proposed studies use yeast and mammalian cells, both eukaryotes in which many aspects of gene regulation are closely related. Among the general issues to be addressed are: how gene 'activators," bound to DNA, communicate with the genes whose transcription is activated; how to design and test small synthetic gene activators; how natural 'activating regions" (peptide sequences that when tethered to DNA interact with the transcriptional machinery to activate transcription) can be replaced by proteins that interact with the transcriptional machinery in a fundamental different, and revealing, fashion; how novel transcriptional activating peptides can be isolated and how they work; how a specific repressor, once tethered to DNA, counteracts the effect of activators; how the appearance of various bits of the transcriptional machinery, at a promoter following activation, can be precisely analyzed; and, in general, how enzymes (e.g. the RNA polymerase responsible for transcribing genes, and a cyclin-dependent kinase found in the transcriptional machinery) are made to function in a specific fashion, selecting one vs another potential target under any specified set of circumstances.